Abstract Genome replication is an essential event in all forms of life. DNA replication is initiated at specific sites (termed origins of replication) along chromosomes to facilitate appropriate duplication of the genome. In humans, the determinants that regulate the location of DNA replication origin are relatively unresolved. Here we will structurally investigate key determinants that establish origins of replication in humans to define the mechanism of DNA origin selection. The initial factor that establishes the origin of replication is the origin recognition complex (ORC). ORC recruits the protein CDC6 to the DNA origin of replication to form the pre- replicative complex (pre-RC). The complex is essential for replication, considering mutations in ORC genes can lead to deleterious effects, such as Meier-Gorlin Syndrome and cancer resulting from incomplete replication. We will investigate ORC?CDC6?DNA interactions through binding studies, such as electrophoretic mobility shift (EMSA) and Frster resonance energy transfer (FRET) assays. The ORC?CDC6?DNA complex will be further analyzed through cryoelectron microscopy (cryo-EM) techniques. Studies have shown ORC and CDC6 are recruited to established locations along variably structured chromatin. The chromatin regions of active transcription consist of histone complexes, called nucleosomes, positioned intermittently along DNA and these nucleosomes influence ORC establishment and therefore replication origin selection. The histone subunits undergo many posttranslational modifications that influence ORC binding to the complex. We will structurally investigate pre-RC?nucleosome interactions through advanced cryo-EM methods to define the first step in genome replication. The pre-RC and nucleosome reconstitution will be optimized to generate stable and homogenous samples. Modern fluorescent labelling-approaches will be developed to analyze the binding properties of the complexes. Posttranslational modifications, such as phosphorylation and methylation, will be addressed to determine the influence on ORC and nucleosome recognition. The results from the structural and binding studies will support development of a ChIP-Seq assay to map out the ORC and replication origin genome location. An in depth understanding of ORC?CDC6?nucleosome interactions are key to unraveling the mechanism of DNA origin selection and will provide insight for the design of pharmaceutical compounds that reverse the effects of incomplete replication. My long-term goal is to become the principal investigator of an independent research laboratory that conducts high impact studies on the structural biology of DNA replication and chromatin regulation. The NIH F32 fellowship will provide immense learning and research support towards this goal. In addition, the Cold Spring Harbor Laboratory harbors national meetings (ex: Eukaryotic DNA Replication & Genome Maintenance), courses (ex: Cryoelectron Microscopy, March), and workshops (Leadership) that are exceptional for my scientific development.